Veterinary surgery : VS | 2021
Toll-like receptor activation of equine mesenchymal stromal cells to enhance antibacterial activity and immunomodulatory cytokine secretion.
Abstract
OBJECTIVE\nTo evaluate effects of Toll-like and nucleotide-binding oligomerization domain (NOD)-like receptor (TLR, NLR) ligand stimulation of equine mesenchymal stromal cells (MSCs) on antibacterial and immunomodulatory properties in vitro.\n\n\nSTUDY DESIGN\nControlled laboratory study.\n\n\nSAMPLE POPULATION\nEquine bone-marrow-derived MSCs (three horses).\n\n\nMETHODS\nMSCs were stimulated with TLR (polyinosinic:polycytidylic acid [pIC] and lipopolysaccharide [LPS]) and NLR agonists (γ-d-Glu-mDAP [IE-DAP]) for 2 h, and plated at 1 ×\u2009105 cells/well 24\u2009h. MSC-conditioned media (MSC-CM) were collected and assessed for antimicrobial peptide cathelicidin/LL-37 production, bactericidal action against multidrug-resistant planktonic and biofilm Staphylococcus aureus and neutrophil phagocytosis. Bacterial growth was measured by plating bacteria and counting viable colonies, reading culture absorbance, and live-dead staining with confocal microscopy imaging. Following initial comparison of activating stimuli, TLR3-agonist pIC protocols (cell density during activation and plating, culture time, %serum) were further optimized for bactericidal activity and secretion of interleukin-8 (IL-8), monocyte-chemoattractant-protein (MCP-1), and cathelicidin/LL37.\n\n\nRESULTS\nMSCs stimulation with pIC (p = .004) and IE-DAP (p = .03) promoted increased bactericidal activity, evidenced by reduced viable planktonic colony counts. PIC stimulation (2 ×\u2009106 cells/ml, 2 h, 10 μg/ml) further suppressed biofilm formation (p = .001), enhanced neutrophil bacterial phagocytosis (p = .009), increased MCP-1 secretion (p\u2009<\u2009.0001), and enhanced cathelicidin/LL-37 production, which was apparent when serum concentration in media was reduced to 1% (p = .01) and 2.5% (p = .05).\n\n\nCONCLUSION\nTLR-3 pIC MSCs activation was most effective to enhance antibacterial and cytokine responses, which were affected by serum reduction.\n\n\nCLINICAL SIGNIFICANCE\nIn vitro TLR-3 activation of equine MSCs tested here may be a strategy to improve antibacterial properties of MSCs to treat antibiotic-resistant infections.